It is generally agreed that hyperlipidemia is a predisposing factor to cardiovascular disease. Lipids are present in the blood serum as lipoproteins. The lipoproteins specifically implicated in cardiovascular disease are classified as low density lipoproteins (LDL) and very low density lipoproteins (VLDL) according to their ultracentrifugal flotation properties, and as beta- and pre-beta lipoproteins, respectively, according to their electrophoretic migration. In addition to the fact that there are various means of classifying lipoproteins, there are a number of recognized methods for isolating and purifying them.
Most lipoprotein purification methods utilize ultracentrifugation either alone or subsequent to a preliminary precipitation of the serum lipoprotein fraction with, e.g. polyanion and a divalent cation. This is a difficult and involved technical operation. The lipoproteins are then classified by one of the methods referred to above. The entire procedure of purifying and classifying lipoproteins is not only difficult but also time consuming.
The quantitative determination of serum beta-lipoproteins is a very valuable medical diagnostic tool. A rapid screening procedure for detecting increased serum beta-lipoprotein levels would be an even more valuable medical diagnostic tool since it would facilitate a means of identifying individuals with occult hyperlipidemia, thereby indicating the need for further and more definitive examination of their blood serum for cholesterol and triglyceride levels. One procedure commonly utilized for determination of serum low density lipoproteins is a turbidimetric estimation. In this method, the turbidity (absorbance) of a sample of serum from an individual who has fasted 18 hours is determined after it has been treated to precipitate the low density beta-lipoproteins by the addition of both a divalent cation, i.e. Ca.sup.++ and a polyanion precipitant. A similar amount of serum is treated with the same volume of reagent containing only the divalent cation. The turbidity (absorbance) of this second solution (reagent blank) is subtracted from the first reading. The turbidity values are expressed in arbitrary absorbance units.
A number of precipitants are described in the art for use in turbidimetric determinations. The preferred reagents for this purpose are polyanions which are utilized in conjunction with a divalent cation. Frequently used polyanions include, for example, dextran sulfate, heparin, amylopectin sulfate and phosphotungstate. Divalent cations commonly utilized include Ca.sup.++, Mg.sup.++, and Mn.sup.++. The procedures utilizing such materials in the turbidimetric determinations are fully described by Burstein and Scholnick: Lipoprotein-polyanion-metal interactions. Adv. Lipid Res. 11. 67 (1973). In this article, Burstein et al mention the use of numerous sulfated polyanions as precipitants for the removal of the beta-lipoprotein fraction from serum. The authors include under this heading sodium polyanethole sulfonate which is incorrect because sodium polyanethole is a polysulfonate as opposed to a polysulfated molecule. In addition, Burstein et al. give no suggestion that sodium polyanethole sulfate could be utilized to prepare a calibration standard which would facilitate the quantitative determination of serum beta-lipoproteins.
The present invention describes the preparation of low- and very low-density serum beta-lipoproteins of immunochemical homogeneity. Beta-lipoproteins prepared in accordance with the invention are useful as calibration standards in the turbidimetric determination of serum beta-lipoproteins. Such determinations performed as described using the calibration standard provided in accordance with the invention have been shown to yield quantitative results and to be, unexpectedly, relatively free from interference from chylomicrons present in the serum.